A kind of polypeptide for ptp1b detection and fluorescent probe comprising the polypeptide

A technology for synthesizing fluorescent probes and solid-phase peptides, applied in the field of fluorescent probes, can solve the problems of large background interference, compound structure influence, and high detection environment requirements in the chromogenic substrate method.

Active Publication Date: 2020-09-29
ZHEJIANG CHINESE MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the chromogenic substrate method has a large background interference and is easily affected by the structure of the compound; the malachite green-molybdate spectrophotometric method has high requirements for the detection environment and cannot directly detect PTP1B activity

Method used

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  • A kind of polypeptide for ptp1b detection and fluorescent probe comprising the polypeptide
  • A kind of polypeptide for ptp1b detection and fluorescent probe comprising the polypeptide
  • A kind of polypeptide for ptp1b detection and fluorescent probe comprising the polypeptide

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Embodiment 1 has the fluorescent probe synthesis of AIE characteristic

[0046] This embodiment provides a method for synthesizing a fluorescent probe with AIE characteristics, and its synthesis process is as follows figure 1 shown, including:

[0047] (1) Separately synthesize a polypeptide chain through a solid-phase polypeptide synthesis reaction, and the sequence of the polypeptide chain is lysine-glutamic acid-phosphorylated tyrosine-phosphorylated tyrosine-lysine-valine;

[0048] (2) Add 4-hydroxybenzophenone (1.9g, 10mmol), benzophenone (2.2g, 12mmol) and zinc powder (2.9g, 44mmol) into a 250ml three-necked flask, pump air, nitrogen, and repeat three times; Add 80ml of THF (tetrahydrofuran), 0 ℃ ice-water bath for 30min; dropwise add titanium tetrachloride (2.4ml, 22mmol) under ice-water bath, reflux overnight, spin dry; add appropriate amount of dichloromethane and dilute hydrochloric acid for extraction, remove the organic layer, dried with anhydrous magnesiu...

Embodiment 2

[0052] Example 2 Application of Fluorescent Probe TPE-KEpYpYKV in PTP1B Activity Detection

[0053] (1) Fluorescence spectrum analysis of the fluorescent probe and the product after the reaction between the fluorescent probe and the enzyme.

[0054] Sample group 1: Add 50 μL of fluorescent probe TPE-KEpYpYKV with a concentration of 100 μM;

[0055] Sample group 2: Add 1.5 μL of PTP1B with a concentration of 40 μg / mL, 50 μL of fluorescent probe TPE-KEpYpYKV with a concentration of 100 μM;

[0056] The two sample groups were buffered with Tris-HCl (10mM, pH 7.5, containing 50mM Na + , 2mMDTT) to 100 μL, and incubated at 37°C for 15 minutes. After the reaction, a Tecan microplate reader was used to measure the fluorescence spectrum from 400 nm to 600 nm under 320 nm excitation.

[0057] See the test results figure 2 .

[0058] Depend on figure 2 It can be seen that the synthesized fluorescent probe TPE-KEpYpYKV basically has no fluorescence absorption in the range of 400-...

Embodiment 3

[0063] Example 3 Application of Fluorescent Probe TPE-KEpYpYKV in Screening PTP1B Inhibitors

[0064] (1) Application of fluorescent probe TPE-KEpYpYKV in screening PTP1B inhibitors

[0065] Take 50 μL of fluorescent probe TPE-KEpYpYKV with a concentration of 100 μM, add 1.5 μL of PTP1B with a concentration of 40 μg / mL and a certain volume of different concentrations of Na 3 VO 4 Mother liquor (making Na 3 VO 4 The final concentration is 0.5, 1, 5, 25, 125, 250, 500μM), with buffer Tris-HCl (10mM, pH 7.5, containing 50mMNa + , 2mMDTT) to 100μL, incubate at 37°C for 15min, measure with a Tecan microplate reader, set E x 320nm (±5nm), E m 480nm (±5nm).

[0066] See the test results Figure 5 .

[0067] Depend on Figure 5 It can be seen that with Na 3 VO 4 Concentration increases, Na 3 VO 4 The inhibitory effect on PTP1B also gradually increased; indicating that the fluorescence growth rate of the fluorescent probe TPE-KEpYpYKV can better reflect the inhibitory effe...

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Abstract

The invention discloses a polypeptide capable of being specifically recognized by PTP1B and a fluorescent probe comprising the polypeptide, and belongs to the field of drug screening and evaluation methods. The fluorescent probe is made of a polypeptide specifically recognized by PTP1B and an aggregation-induced luminescent molecule through connection. The amino acid sequence of the polypeptide is: lysine-glutamate-phosphotyrosine-phosphotyrosine-lysine-valine. The aggregation-induced luminescent molecule is linked to the lysine in the polypeptide. Since the third and fourth positions at N terminal of the polypeptide contain the phosphotyrosine, the PTP1B can modify the polypeptide by dephosphorylation. The fluorescent probe has substantially no fluorescence absorption, but can produce significant fluorescence absorption when the fluorescent probe loses phosphate on the tyrosine, so as to specifically detect the activity of PTP1B.

Description

technical field [0001] The invention belongs to the field of drug screening and evaluation methods, and specifically relates to a polypeptide that can be specifically recognized by PTP1B and a fluorescent probe containing the polypeptide. Background technique [0002] Protein tyrosine phosphorylation and dephosphorylation is one of the body's signal transduction regulatory mechanisms, which are jointly regulated by protein tyrosine kinases and protein tyrosine phosphatases. Protein Tyrosine Phosphatase-1B (Protein Tyrosine Phosphatase-1B, PTP1B) is a member of the intracellular protein tyrosine phosphatase family, located in the endoplasmic reticulum of cells, and widely expressed in liver, muscle, brain, fat and other tissues. Studies have shown that PTP1B is a common negative regulator of leptin signaling pathway and insulin signaling pathway. PTP1B blocks insulin and leptin signaling by dephosphorylating insulin receptors, insulin receptor substrates, and Janus kinase 2 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06C07K1/13C09K11/06G01N33/573
CPCC07K7/06C09K11/06C09K2211/1007C09K2211/1014G01N33/573G01N2333/916
Inventor 赵筱萍王毅邹敬韬
Owner ZHEJIANG CHINESE MEDICAL UNIVERSITY
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